PROJECT SUMMARY Glaucoma represents a group of diseases, which are associated with multiple risk factors and genetic variants. The unifying theme among these diseases is the progressive degeneration of optic nerve and retinal ganglion cells (RGCs) degenerate, leading to irreversible blindness. Based on this observation we propose a hypothesis that RGCs are intrinsically vulnerable to glaucoma risk factors and genetic variants. Since RGCs are born embryonically and glaucoma is an adult onset disease our knowledge about the emergence of and mechanism underlying RGC susceptibility, important for early diagnosis and formulating therapeutic approaches, remain rudimentary. Our objective is to examine the impact of genetic variations associated with glaucoma on the development, phenotype, and regeneration of RGCs, using the induced pluripotent stem cell (iPSC) model of primary open angle glaucoma (POAG), the most prevalent type of the disease. Here, we will test the hypothesis that RGCs in the SIX6 risk allele (Asn141His, rs33912345) in POAG are developmentally compromised in normal phenotype and function, and that this affects their survival and regeneration. The SIX6 variant is a suitable target for the analysis because SIX6 is one of the eye-field genes involved in retinal development and the missense mutation (Asn141His) in the DNA binding region of SIX6 is accompanied by degenerative changes that include reduction in the retinal nerve fiber layer (RNFL) thickness in POAG. Thus, the generation of SIX6 risk allele-POAG RGCs in a dish model of the disease would allow the characterization of developmental and phenotype abnormalities, shedding light on glaucomatous RGC susceptibility. We have proposed three aims to test the central hypothesis. First, we will determine the impact of the SIX6 risk allele-POAG on the development and phenotype of patient-specific RGCs by systematic characterization of sequential steps of retinal inductions and RGC differentiation of POAG patient-specific and age- and sex-matched control iPSCs, under the influence of a stage-specific and chemically defined protocol. Second, we will examine the impact of the SIX6 risk allele-POAG on patient-specific RGC axon growth and regeneration in the context of mTOR signaling, a regulator of retinal development and regeneration, which is inhibited in POAG patient- specific RGCs. Lastly, we will identify the molecular pathway(s) underlying abnormal RGC development and phenotype. We will carry out hypothesis-driven (e.g., HMGA2 and KLF-4 as candidate hub genes) genome wide transcriptional analysis of POAG patient-specific RGCs during development and regeneration to understand the molecular mechanism underlying SIX6 risk allele associated RGC abnormalities. Information emerging from our study will bridge a gap in our knowledge of the intrinsic vulnerability of RGCs in glaucoma. The information on dysregulated processes and pathways will reveal biomarkers for early diagnosis, and will allow strategies for therapeutic regeneration of glaucomatous RGCs.